The actin remodeling protein cofilin is crucial for thymic αβ but not γδ T-cell development

<div><p>Cofilin is an essential actin remodeling protein promoting depolymerization and severing of actin filaments. To address the relevance of cofilin for the development and function of T cells in vivo, we generated knock-in mice in which T-cell–specific nonfunctional (nf) cofilin was expressed instead of wild-type (WT) cofilin. Nf cofilin mice lacked peripheral αβ T cells and showed a severe thymus atrophy. This was caused by an early developmental arrest of thymocytes at the double negative (DN) stage. Importantly, even though DN thymocytes expressed the TCRβ chain intracellularly, they completely lacked TCRβ surface expression. In contrast, nf cofilin mice possessed normal numbers of γδ T cells. Their functionality was confirmed in the γδ T-cell–driven, imiquimod (IMQ)-induced, psoriasis-like murine model. Overall, this study not only highlights the importance of cofilin for early αβ T-cell development but also shows for the first time that an actin-binding protein is differentially involved in αβ versus γδ T-cell development.</p></div

Mice expressing nf cofilin show a severe thymus atrophy and a developmental arrest at the DN3 stage.

<p>(A) Total T-cell number in spleen of B6 mice and nf cofilin knock-in mice (<i>n</i> = 6 independent experiments with a total of ≥8 mice per group). (B) Thymus was isolated and weighed, and the total cell number was determined from 4–5-weeks-old B6 or nf cofilin knock-in mice (<i>n</i> = 6 independent experiments with a total of ≥10 mice per group). (C) Flow cytometric analysis of thymocyte differentiation by CD4, CD8, CD25, and CD44 staining (<i>n</i> ≥ 8 mice per group). Exemplary dot blots from representative mice are shown on the left, whereas the statistical evaluation of summary data is shown in the middle (for DN, DP, and SP stages) and on the right (for DN cell stages). (D) Creation of mixed bone marrow chimera. Lethally irradiated B6 mice were reconstituted with equal numbers of CD3⁺ cell–depleted BM cells from CD45.2⁺ tester (Cfl1^nf/nf) and CD45.1⁺ competitor (B6) mice. Total chimerism was measured and CD4 versus CD8 plots show the developmental stage of thymocytes derived from CD45.1⁺ or CD45.2⁺ BM cells. Plots are representative of six mixed chimeras per group. Bar graphs show the average abundance of each major thymocyte population within the chimera from both tester (CD45.2⁺) and competitor (CD45.1⁺) donor cells. Data is represented as mean ± SEM. **** = p<0.0001; ** = p<0.01. Underlying Data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005380#pbio.2005380.s006" target="_blank">S1 Data</a>. BM, bone marrow; DN, double negative; DP, double positive; SP, single positive.</p

DN thymocytes of Cfl1^nf/nf mice show a dramatically enhanced F-actin content and impaired migratory capacity as well as a lack of TCRβ surface expression.

<p>(A) CD3⁺ splenocytes were analyzed for expression of TCRβ and TCRγδ. Shown are representative dot blots (left panels) and calculated absolute cell numbers (right panel) of TCRβ and TCRγδ expressing cells. Data is represented as mean ± SEM and summarizes 3 independent experiments with a total of 6 mice per group. (B) Total F-actin amount of DN thymocytes or γδ thymocytes was determined by SiR-actin staining (<i>n</i> = 3 independent experiments with a total ≥6 mice per group). (C) Migratory capacity of DN cells or γδ thymocytes was determined in a transwell assay (pore size 5 μm) in which SDF-1α (200 ng/ml) was used as chemotactic stimulus. Migration was carried out for 3 h (<i>n</i> = 3 independent experiments with ≥4 mice per group). (D) TCRβ surface (surface TCRβ) and intracellular (ic TCRβ) expression was analyzed in DN cells by flow cytometry. Representative dot plots from TCRβ versus TCRγδ staining on B6 and Cfl1^nf/nf DN cells are shown (<i>n</i> = 4 independent experiments with a total of ≥7 mice per group). (E) Analysis of surface and ic expression of TCRβ in DN cells of B6 (grey bar) and Cfl1^nf/nf mice (black bar) (left bar chart). Analysis of MFI of TCRβ of icTCRβ⁺ DN cells of B6 (grey bar) and Cfl1^nf/nf mice (black bar) (right bar chart). (F) Analysis of surface expression of TCRβ in DN thymocytes of B6 and Cfl1^nf/nf mice before (-cytoD) and after cytochalasin D treatment (+cytoD). Data is represented as mean ± SEM. **** <i>p</i> < 0.0001; *** <i>p</i> < 0.001; ** <i>p</i> < 0.01; * <i>p</i> < 0.05; Underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005380#pbio.2005380.s006" target="_blank">S1 Data</a>. cytoD, cytochalasin D; ic, intracellular; MFI, mean fluorescence intensity nf, nonfunctional; ns, not significant.</p

Cfl1^nf/nf mice show normal γδ T-cell subsets which remain functional.

<p>(A) Analysis of the surface expression of Vγ1, Vγ2, and Vγ3 of γδ T-cells isolated from the spleen, skin, lung, and thymus of B6 (grey bar) or Cfl1^nf/nf mice (black bar) (<i>n</i> = 4 independent experiments with ≥4 mice per group). (B) Analysis of the surface expression of CD24, CD27, and CD44 of γδ thymocytes of B6 (grey bar) or Cfl1^nf/nf mice (black bar) (<i>n</i> = 4 independent experiments with ≥4 mice per group). (C) In vitro activation of splenic γδ T cells of Cfl1^nf/nf (black bar) and control mice (grey bar) by plate-bound CD3 and CD28 antibodies for 24 h. Determination of the T-cell activation markers CD25 (left bar chart) and CD69 (right bar chart) by flow cytometry. (D) Age- and sex-matched Cfl1^nf/nf (red line) and Cfl1^+/+ (black line) mice at 7 weeks of age were used for an IMQ-induced psoriasis-like model. Over 6 days, prior to topical application of IMQ, scores of individual parameters such as scaling, back skin thickness, and erythema formation were measured and the accumulated PASI was calculated. (E) Flow cytometric analysis of IL-17A and RORγt expression in γδ T cells of skin-draining LNs. Cytokine production was assessed after 6 days of topical application of IMQ containing Aldara crème (Sham) or control crème (Aldara) (experiment with ≥4 mice per group). Data are represented as mean ± SEM. **** <i>p</i> < 0.0001; *** <i>p</i> < 0.001; ** <i>p</i> < 0.01; * <i>p</i> < 0.05; Underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005380#pbio.2005380.s006" target="_blank">S1 Data</a>. Cfl1, cofilin-1; IMQ, imiquimod; ns, not significant; PASI, psoriasis area severity index.</p

IL-7R signaling protects Rag⁻ mice from DSS-induced colitis.

<p>(<b>A, B</b>) WT (n = 4), Rag⁻ (n = 8), Rag⁻IL-7R⁻ (n = 7) and Rag⁻OT-I⁺ mice (n = 6) received dextran sulfate sodium (DSS) via the drinking water. From day 5 on, DSS-free drinking water was provided. (<b>A</b>) Body weight was determined every day and calculated in relation to the initial body weight. Shown are the mean relative body weight ± SEM and the time after onset of DSS treatment. (<b>B, C</b>) Colon samples were taken at day 8 and analyzed histologically. Shown are histological scores for groups of untreated (open symbols; n = 4) and DSS-treated mice (closed symbols; n = 6–8). (<b>C</b>) Shown are representative colon sections from the indicated mice. (<b>A–C</b>) Data represent one experiment.</p

IL-7 promotes IEC proliferation and survival.

<p>(<b>A, B</b>) Rag⁻IL-7⁻ (IL-7⁻; n = 3) and Rag⁻IL-7R⁻ (IL-7R⁻; n = 3) mice were treated with PBS (white bars) or IL-7/anti-IL-7 (black bars) twice a week for 2 weeks. (<b>A, C</b>) Colon wall thickness (µm) and (<b>B, D</b>) the percentage of Ki67⁺ cells in crypts were determined in colon sections from (<b>A, B</b>) IL-7-treated Rag⁻IL-7⁻ and Rag⁻IL-7R⁻ mice and (<b>C, D</b>) untreated WT (n = 6) and IL-7tg (n = 5) mice. (<b>A</b>) 30–54, (<b>B</b>) 15–23, (<b>C</b>) 47–68 and (<b>D</b>) 29–32 individual measurements were performed per experimental group. Shown are mean values+SEM. Statistically significant values are indicated: * p<0.05 and ** p<0.01 (Student's t test). (<b>E</b>) Colon sections from PBS-treated (upper row) and IL-7/anti-IL-7-treated Rag⁻IL-7⁻ (lower row; n = 3) were stained with DAPI and antibodies for Ki67, EpCam, cleaved caspase 3 (Casp3) or β-catenin (βcat). (<b>F</b>) Colon sections from WT (n = 5) and IL-7tg mice (n = 6) were stained with DAPI and antibodies for β-catenin. (<b>E, F</b>) White arrows indicate nuclei. Bar diagrams show the percentage of luminal IEC with nuclear β-catenin. 130–280 nuclei per experimental group were counted. Shown are mean values+SEM. Statistically significant values are indicated: * p<0.05 and ** p<0.01 (Student's t test). (<b>A–F</b>) Data represent one experiment with a total of 23 individual mice and 2–3 independent staining reactions per mouse.</p

IL-7R signaling promotes lymphopenia-associated IEC hyperplasia and alters colon function.

<p>(<b>A</b>) Colon sections from WT (n = 13–16), Rag⁻ (n = 16–37) and Rag⁻IL-7R⁻ mice (n = 13) were stained with DAPI and antibodies for Ki67, EpCam, cleaved caspase 3 (Casp3) or Gob5. (<b>B</b>) Colon wall thickness (µm) and (<b>C</b>) the percentage of Ki67⁺ cells in crypts were determined for WT (n = 6), Rag⁻ (n = 12) and Rag⁻IL-7R⁻ (n = 6) mice. (<b>B, C</b>) 30–60 individual measurements were performed. (<b>A–C</b>) Data are representative for 4 independent experiments and 2–3 staining reactions per mouse. (<b>D</b>) Transepithelial resistance (Ω·cm²), and apparent permeabilities (P) for (<b>E</b>) Na⁺ and (<b>F</b>) Cl⁻ were determined for colon samples from WT (n = 6), Rag⁻ (n = 5) and Rag⁻IL-7R⁻ (n = 5) mice. Five to twelve independent measurements per experimental group were performed. (<b>B–F</b>) Shown are mean values+SEM. Statistically significant values are indicated: ** p<0.01 and *** p<0.001 (Student's t test).</p

Elevated levels of IL-7 expression and IEC hyperplasia in the colon of Rag⁻ mice.

<p>(<b>A</b>) Representative bioluminescence (BL) images for Rag-competent (Rag⁺; n = 31) and Rag-deficient IL-7GCDL mice (Rag⁻; n = 21) are shown. BL was determined (<b>B</b>) in the intestine and (<b>C</b>) thymus, heart, lung, liver, skin and kidney of Rag⁺ (n = 12) and Rag⁻ IL-7GCDL mice (n = 12). (<b>A–C</b>) BL is shown in photons per s per cm² per steradian. (<b>D, E</b>) Colon sections from Rag⁺ (n = 5–8) and Rag⁻ IL-7GCDL mice (n = 6–8) were stained with (<b>D</b>) periodic acid-Schiff (PAS)/Alcian blue (AB) or (<b>E</b>) DAPI and antibodies for IL-7 and EpCam. (<b>D</b>) Differentiated goblet cells stain positive for PAS (red) and appear purple/magenta. Acidic mucopolysaccharides/glycosaminoglycans are visualized by AB. Arrows indicate the distance between the basis of the crypts and the colon lumen. (<b>D, E</b>) Data are representative for 3 independent experiments and 2–3 staining reactions per mouse.</p

IEC express functional IL-7R.

<p>(<b>A, B</b>) IEC were isolated from the colon of (<b>A</b>) Rag⁻IL-7R⁻ and (<b>A, B</b>) Rag⁻ mice. (<b>B</b>) Rag⁻ IEC were stimulated with 20 ng/ml rec. IL-7 for 15 minutes or were left untreated. The levels of IL-7R expression and Stat5 phosphorylation (p-Stat5) were determined by flow cytometry. (<b>A, B</b>) Shown are relative cell numbers and relative fluorescence intensities for (<b>A</b>) IL-7R and (<b>B</b>) p-Stat5 after gating on viable (<b>A</b>) (7AAD⁻), (<b>A, B</b>) CD45⁻, EpCam⁺ IEC. Results are representative for 2–3 independent experiments.</p

Peripheral inflammation increases the number of recombined Purkinje neurons.

<p>The number of recombined Purkinje neurons is low in healthy animals (A) but increases dramatically after peripheral inflammatory conditions (B). Inflammatory injuries were induced by subcutaneous injection of LLC2s or peritonitis. Animals were analyzed 12 d after injection when tumors were formed. Peritonitis was induced by a single i.p. injection of thioglycolate broth (1 ml in 3% PBS). Mice with peritonitis and ECL were analyzed 4 d after injection. (C) Filled bars represent results from Vav-iCre and empty bars from Tie2-Cre reporter mice. The <i>p</i> values were calculated by two-tailed <i>t</i> test for groups with unequal variance. (D and E) We did not observe any recombined Purkinje neurons that were binucleated in either transgenic mouse line after induction of an inflammation. (F and G) Microglia (white arrows) were always negative for the marker gene in healthy animals as well as after an inflammation. (H and I) Transendothelial electrical resistance (TEER, top panel) decreases and the corresponding capacitance (Ccl, bottom panel) of the bEnd5 endothelial monolayers increases significantly 24 h and 48 h after addition of bone-marrow-derived EVs compared to conditioned medium supernatant after ultracentrifugation. Vertical line at 0 h indicates media exchange. Scale bar, 100 µm (A and B), 50 µm (D and F), 10 µm (E), and 5 µm (G).</p